Cells and tissues establish and maintain their unique architectures in large part through the tight regulation of protein and membrane transport. One key aspect of this process is endocytic recycling, the selective return of internalized macromolecules to the cell surface from endosomes. Understanding endocytic recycling is of fundamental importance to cell biology and has broad relevance to many areas of biomedicine including cancer and type II diabetes. Our general approach has been to exploit powerful features of C. elegans genetics to characterize proteins that are required for the recycling process in vivo. We then extend these findings to mammalian cells and to in vitro analysis of the relevant C. elegans proteins and their mammalian homologs. For many of these studies we focused on a system that we pioneered, the C. elegans intestine, a very simple model that allows facile analysis of endocytic membrane transport pathways within intact polarized epithelia. During the previous granting period we gained new understanding of how RME-1/EHD family proteins, identified in our previous screens, function in endosomal membrane tubulation and fission, and identified new proteins that function with RME-1 in this process. We also identified and characterized several new effectors for the small GTPase RAB-10/Rab10, a protein that we previously showed is a master regulator of basolateral recycling in the worm intestine and polarized mammalian MDCK cells. We propose three new aims to further elucidate the molecular mechanisms underlying endocytic recycling. We plan to decipher a novel inhibitory cascade regulating early endosome GTPase RAB-5 during receptor recycling. New data indicates regulation by recycling endosome proteins RAB-10, CED-10/Rac, and AMPH-1/Amphiphysin. We will also elucidate the molecular links of newly identified RAB-10 effector EHBP-1 to membranes, the cytoskeleton, and apparent feedback regulation of RAB-10. Finally, we focus on understanding the molecular basis of endosomal regulation by a new player in the RAB-10 pathway, putative ARF6 effector UNC-16/ JIP3. We connect UNC-16 to an ARF-GAP in this pathway and to a new Rab-GTPase regulator. The experiments proposed here should provide significant new insights into how endocytic recycling works. Given the high level of phylogenetic conservation of such pathways from worms to mammals, and our parallel analysis in human cells, we expect that our work will provide extensive insight into key conserved elements relevant across species. This work is important for understanding disease etiology and in identifying therapeutic targets for disease intervention. PUBLIC HEALTH RELEVANCE: Our research focuses on the return of internalized macromolecules to the cell surface from endosomes (endocytic recycling), a basic cell biological process that is of fundamental importance to many areas of biomedicine. For instance, endocytic recycling is a key control point in the insulin-stimulated movement of glucose transporters (Glut4) from endosomes to the plasma membrane, a process that goes awry in type II diabetes. A better understanding of how endocytic recycling functions will be profoundly important in identifying therapeutic targets to combat this and other diseases.